Methods, cells and reagents for production of isoprene, derivatives and intermediates thereof

ABSTRACT

This application describes methods, including non-naturally occurring methods, for biosynthesizing 3-hydroxy-3-methylglutaryl-coA and intermediates thereof, as well as non-naturally occurring hosts for producing 3-hydroxy-3-methylglutaryl-coA. This application also describes methods, including non-naturally occurring methods, for biosynthesizing isoprene and intermediates thereof, as well as non-naturally occurring hosts for producing isoprene.

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/205,926, filed Aug. 17, 2015.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 26, 2016, isnamed 12444_0582-00000_SL.txt and is 31,431 bytes in size.

TECHNICAL FIELD

This application relates to methods for biosynthesizing3-hydroxy-3-methylglutaryl-coA (3-HMG) and intermediates thereof, usingone or more isolated enzymes such as one or more of a4-methyl-2-oxopentanoate, a 3-methylbutanoyl-CoA oxidoreductase, a3-methylbut-2-enoyl-CoA carboxylase, and a 3-methylglutaconyl-CoAhydratase; or using non-naturally occurring host cells expressing one ormore such enzymes.

This application further relates to methods for biosynthesizing isopreneand intermediates thereof from 3-hydroxy-3-methylglutaryl-coA using oneor more isolated enzymes, such as one or more of a hydroxymethylglutarylCo-A reductase, a mevalonate-kinase, a phosphomevalonate kinase, adiphosphomevalonate decarboxylase, an isopentenyl diphosphate isomerase,and an isoprene synthase; or using non-naturally Occurring host cellsexpressing one or more such enzymes.

BACKGROUND

Isoprene is an important monomer for the production of specialtyelastomers including motor mounts/fittings, surgical gloves, rubberbands, golf balls and shoes. Styrene-isoprene-styrene block copolymersform a key component of hot-melt pressure-sensitive adhesiveformulations and cis-poly-isoprene is utilized in the manufacture oftires (Whited et al., Industrial Biotechnology, 2010, 6(3), 152-163).

Manufacturers of rubber goods depend on either imported natural rubberfrom the Brazilian rubber tree or petroleum-based synthetic rubberpolymers (Whited et al., 2010, supra). Given a reliance on petrochemicalfeedstocks and the harvesting of trees, biotechnology offers analternative approach via biocatalysis. Biocatalysis is the use ofbiological catalysts, such as enzymes, to perform biochemicaltransformations of organic compounds.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing intermediates, in particularisoprene, wherein the methods are biocatalysis based.

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes. The introduction ofvinyl groups into medium carbon chain length enzyme substrates is a keyconsideration in synthesizing isoprene via biocatalysis processes.

There are known metabolic pathways leading to the synthesis of isoprenein prokaryotes such as Bacillis subtillis and eukaryotes such as Populusalba (Whited et al., 2010, supra).

Isoprene may be synthesized via two routes leading to the precursordimethylvinyl-PP, such as the mevalonate and the non-mevalonate pathway(Kiiztiyama, Biosci. Biotechnol. Biochem., 2002, 66(8), 1019-1827).

The mevalonate pathway incorporates a decarboxylase enzyme, mevalonatediphosphate decarboxylase (hereafter Mdd), that introduces the firstvinyl-group into the precursors leading to isoprene. The secondvinyl-group is introduced by isoprene synthase (hereafter IspS) in thefinal step in synthesizing isoprene.

The mevalonate pathway (shown in part in FIG. 2) has been exploited inthe biocatalytic production of isoprene using E. coli as host. E. coliengineered with the mevalonate pathway requires three moles ofacetyl-CoA, three moles of ATP and two moles of NAD(P)H to produce amole of isoprene. Given a theoretical maximum yield of 25.2% (w/w) forthe mevalonate pathway, isoprene has been produced biocatalytically at avolumetric productivity of 2 g/(L·h) with a yield of 11% (w/w) fromglucose (Whited et al., 2010, supra). Particularly, the phosphateactivation of mevalonate to 5-diphosphomevalonate is energy intensivemetabolically, requiring two moles of ATP per mole of isoprene synthesis(FIG. 2). Accordingly, reducing the ATP consumption can improve theefficiency of the pathway.

SUMMARY

The inventors have determined that it is possible to biosynthesize 3-HMGand/or intermediates thereof from 4-methyl-2-oxopentanoate using one ormore isolated enzymes, or using non-naturally occurring host cellsexpressing one or more such enzymes. For example, 3-HMG may bebiosynthesized from 4-methyl-2-oxopentanoate using one or more of a4-methyl-2-oxopentanoate dehydrogenase, a 3-methylbutanoyl-CoAoxidoreductase, a 3-methylbut-2-enoyl-CoA carboxylase, and a3-methylglutaconyl-CoA hydratase. For further example, 3-HMG may bebiosynthesized from 4-methyl-2-oxopentanoate using one or more of a4-methyl-2-oxopentanoate decarboxylase, a 3-methylbutanal dehydrogenase,a 3-methylbutanoate-CoA ligase, a 3-methylbutanoyl-CoA oxidoreductase, a3-methylbut-2-enoyl-CoA carboxylase, and a 3-methylglutaconyl-CoAhydratase.

In one embodiment, are methods, including non-naturally occurringmethods, for synthesizing 3-HMG, comprising enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA using a polypeptidehaving the activity of an EC 1.2.7.7 or EC 1.2.1.- enzyme, enzymaticallyconverting 3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA using apolypeptide having the activity of an EC 1.3.8.4 enzyme, enzymaticallyconverting 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl- using apolypeptide having the activity of an EC 6.4.1.4 enzyme, andenzymatically converting 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA using a polypeptide having the activityof an EC 4.2.1.18 enzyme.

In one embodiment, are methods, including non-naturally occurringmethods, for synthesizing 3-HMG, comprising enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanal using a polypeptide havingthe activity of an EC 4.1.1.74 or EC 4.1.1.43 enzyme, enzymaticallyconverting 3-methylbutanal to 3-methylbutanoate using a polypeptidehaving the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme,enzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA usinga polypeptide having the activity of an EC 6.2.1.2 enzyme, enzymaticallyconverting 3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA using apolypeptide having the activity of an EC 1.3.8.4 enzyme, enzymaticallyconverting 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl- using apolypeptide having the activity of an EC 6.4.1.4 enzyme, andenzymatically converting 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA using a polypeptide having the activityof an EC 4.2.1.18 enzyme.

The inventors have also determined that it is possible to biosynthesizeisoprene and/or intermediates thereof from 4-methyl-2-oxopentanoate viaa 3-HMG intermediate using one or more isolated enzymes, or usingnon-naturally occurring host cells expressing one or more such enzymes.For example, isoprene may be synthesized from 4-methyl-2-oxopentanoateusing one or more of a 4-methyl-2-oxopentanoate dehydrogenase, a3-methylbutanoyl-CoA oxidoreductase, a 3-methylbut-2-enoyl-CoAcarboxylase, a 3-methylglutaconyl-CoA hydratase, a hydroxymethylglutarylCo-A reductase, a mevalonate-kinase, a phosphomevalonate kinase, adiphosphomevalonate decarboxylase, an isopentenyl diphosphate isomerase,and an isoprene synthase. For further example, isoprene may bebiosynthesized from 4-methyl-2-oxopentanoate using one or more of a4-methyl-2-oxopentanoate decarboxylase, a 3-methylbutanal dehydrogenase,a 3-methylbutanoate-CoA ligase, a 3-methylbutanoyl-CoA oxidoreductase, a3-methylbut-2-enoyl-CoA carboxylase, a 3-methylglutaconyl-CoA hydratase,a hydroxymethylglutaryl Co-A reductase, a mevalonate-kinase, aphosphomevalonate kinase, a diphosphomevalonate decarboxylase, anisopentenyl diphosphate isomerase, and an isoprene synthase.

In one embodiment, are methods, including non-naturally occurringmethods, for synthesizing isoprene via a 3-HMG intermediate, comprisingenzymatically converting 4-methyl-2-oxopentanoate to3-methylbutanoyl-CoA using a polypeptide having the activity of an EC1.2.7.7 or EC 1.2.1.- enzyme, enzymatically converting3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA using a polypeptidehaving the activity of an EC 1.3.8.4 enzyme, enzymatically converting3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl- using a polypeptidehaving the activity of an EC 6.4.1.4 enzyme, enzymatically converting3-methyl-glutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA using apolypeptide having the activity of an EC 4.2.1.18 enzyme, enzymaticallyconverting 3-hydroxy-3-methylglutaryl-CoA to (R)-mevalonate using apolypeptide having the activity of an EC 1.1.1.34 enzyme, enzymaticallyconverting (R)-mevalonate to (R)-5-phosphomevalonate using a polypeptidehaving the activity of an EC 2.7.1.36 enzyme, enzymatically converting(R)-5-phosphomevalonate to (R)-5-diphosphomevalonate using a polypeptidehaving the activity of an EC 2.7.4.2 enzyme, enzymatically converting(R)-5-diphosphomevalonate to isopentenyl diphosphate using a polypeptidehaving the activity of an EC 4.1.1.33 enzyme, enzymatically convertingisopentenyl diphosphate to dimethylallyl diphosphate using a polypeptidehaving the activity of an EC 5.3.3.2 enzyme, and enzymaticallyconverting dimethylallyl diphosphate to isoprene using a polypeptidehaving the activity of an EC 4.2.3.27 enzyme.

In one embodiment, are methods, including non-naturally occurringmethods, for synthesizing isoprene via a 3-HMG intermediate, comprisingenzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanalusing a polypeptide having the activity of an EC 4.1.1.74 or EC 4.1.1.43enzyme, enzymatically converting 3-methylbutanal to 3-methylbutanoateusing a polypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5enzyme, enzymatically converting 3-methylbutanoate to3-methylbutanoyl-CoA using a polypeptide having the activity of an EC6.2.1.2 enzyme, enzymatically converting 3-methylbutanoyl-CoA to3-methylbut-2-enoyl-CoA using a polypeptide having the activity of an EC1.3.8.1 enzyme, enzymatically converting 3-methylbut-2-enoyl-CoA to3-methyl-glutaconyl- using a polypeptide having the activity of an EC6.4.1.4 enzyme, enzymatically converting 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA using a polypeptide having the activityof an EC 4.2.1.18 enzyme, enzymatically converting3-hydroxy-3-methylglutaryl-CoA to (R)-mevalonate using a polypeptidehaving the activity of an EC 1.1.1.34 enzyme, enzymatically converting(R)-mevalonate to (R)-5-phosphomevalonate using a polypeptide having theactivity of an EC 2.7.1.36 enzyme, enzymatically converting(R)-5-phosphomevalonate to (R)-5-diphosphomevalonate using a polypeptidehaving the activity of an EC 2.7.4.2 enzyme, enzymatically converting(R)-5-diphosphomevalonate to isopentenyl diphosphate using a polypeptidehaving the activity of an EC 4.1.1.33 enzyme, enzymatically convertingisopentenyl diphosphate to dimethylallyl diphosphate using a polypeptidehaving the activity of an EC 5.3.3.2 enzyme, and enzymaticallyconverting dimethylallyl diphosphate to isoprene using a polypeptidehaving the activity of an EC 4.2.3.27 enzyme.

In one embodiment, are methods, including non-naturally occurringmethods, for synthesizing isoprene via a 3-HMG intermediate, comprisingenzymatically converting 3-hydroxy-3-methylglutaryl-CoA to(R)-mevalonate using a hydroxymethylglutaryl Co-A reductase enzyme, forexample a hydroxymethyiglutaryl Co-A reductase having the amino acidsequence set forth in SEQ ID No: 1 or a functional fragment thereof;enzymatically converting (R)-mevalonate to (R)-5-phosphomevalonate usinga mevalonate-kinase enzyme, for example a mevalonate-kinase having theamino acid sequence set forth in SEQ ID No: 2 or a functional fragmentthereof; enzymatically converting (R) 6 phosphomevalonate to(R)-5-diphosphomevalonate using a phosphomevalonate kinase enzyme, forexample a phosphomevalonate kinase having the amino acid sequence setforth in SEQ ID No: 3 or a functional fragment thereof; enzymaticallyconverting (R)-5-diphosphomevalonate to isopentenyl diphosphate using adiphosphomevalonate decarboxylase enzyme, for example adiphosphomevalonate decarboxylase having the amino acid sequence setforth in SEQ ID No: 4 or a functional fragment thereof, or adiphosphomevalonate decarboxylase having the amino acid sequence setforth in SEQ ID No: 5 or a functional fragment thereof; enzymaticallyconverting isopentenyl diphosphate to dimethylallyl diphosphate using anisopentenyl diphosphate isomerase, for example an isopentenyldiphosphate isomerase having the amino acid sequence set forth in SEQ IDNo: 6 or a functional fragment thereof; and enzymatically convertingdimethylallyl diphosphate to isoprene using an isoprene synthase enzyme,for example an isoprene synthase having the amino acid sequence setforth in SEQ ID No: 7 or a functional fragment thereof.

In one embodiment, the methods for synthesizing 3-HMG from4-methyl-2-oxopentanoate and for synthesizing isoprene from4-methyl-2-oxopentanoate via a 3-HMG intermediate are performed in anon-naturally occurring host, which may be a prokaryotic or eukaryotichost.

In one embodiment, at least one of the enzymatic conversions within themethods for synthesizing 3-HMG from 4-methyl-2-oxopentanoate and forsynthesizing isoprene from 4-methyl-2-oxopentanoate via a 3-HMGintermediate is performed in a non-naturally occurring host, which maybe a prokaryotic or eukaryotic host.

In one embodiment, are non-naturally occurring hosts capable ofsynthesizing 3-HMG from 4-methyl-2-oxopentanoatc, said host comprisingat least One exogenous nucleic acid encoding a polypeptide having theactivity of an EC 1.2.7.7 or EC 1.2.1.- enzyme, at least one exogenousnucleic acid encoding a polypeptide having the activity of an EC 1.3.8.4enzyme, at least one exogenous nucleic acid encoding a polypeptidehaving the activity of an EC 6.4.1.4 enzyme; and at least one exogenousnucleic acid encoding a polypeptide having the activity of an EC4.2.1.18 enzyme.

In one embodiment, are non-naturally occurring hosts capable ofsynthesizing 3-HMG from 4-methyl-2-oxopentanoate, said host comprisingat least one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 4.1.1.74 or EC 4.1.1.43 enzyme, at least one exogenousnucleic acid encoding a polypeptide having the activity of an EC1.2.1.39 or EC 1.2.1.5 enzyme, at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 6.2.1.2. enzyme, atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 1.3.8.4 enzyme, at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 6.4.1.4 enzyme, andat least one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 4.2.1.18 enzyme.

In one embodiment, are non-naturally occurring hosts capable ofsynthesizing 3-HMG from 4-methyl-2-oxopentanoate via both of thepathways disclosed above. In one embodiment, are non-naturally occurringhosts capable of synthesizing 3-HMG from 4-methyl-2-oxopentanoate viasimultaneous operation of both of the pathways disclosed above.

In one embodiment, are non-naturally occurring hosts capable ofsynthesizing isoprene from 4-methyl-2-oxopentanoate via a 3-HMGintermediate, said host comprising at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 1.2.7.7 or EC1.2.1.- enzyme, at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 1.3.8.4 enzyme, at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 6.4.1.4 enzyme, at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 4.2.1.18 enzyme, at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 1.1.1.34 enzyme, at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 2.7.1.36 enzyme, at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 2.7.4.2 enzyme, at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 4.1.1.33 enzyme, at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 5.3.3.2 enzyme, and least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 4.2.3.27 enzyme.

In one embodiment, are non-naturally occurring hosts capable ofsynthesizing isoprene from 4-methyl-2-oxopentanoate via a 3-HMGintermediate, said host comprising at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 4.1.1.74 or EC4.1.1.43 enzyme, at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme,at least one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 6.2.1.2. enzyme, at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 1.3.8.4 enzyme, atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 6.4.1.4 enzyme, at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 4.2.1.18 enzyme, atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 1:1.1.34 enzyme, at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 2.7.1.36 enzyme, atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 2.7.42 enzyme, at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 4.1.1.33 enzyme, atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 5.3.3.2 enzyme, and least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 4.2.3.27 enzyme.

In one embodiment, are non-naturally occurring hosts capable ofsynthesizing isoprene from 4-methyl-2-oxopentanoate via a 3-HMGintermediate via both of the pathways disclosed above. In oneembodiment, are non-naturally occurring hosts capable of synthesizingisoprene from 4-methyl-2-oxopentanoate via a 3-HMG intermediate viasimultaneous operation of both of the pathways disclosed above.

In one embodiment, hosts may be capable of endogenously producingisoprene, for example via a non-mevalonate pathway.

In one embodiment, at least one of the enzymatic conversions of themethods comprises gas fermentation, for example fermentation of at leastone of natural gas, syngas, CO₂/H₂, methanol, ethanol, non-volatileresidue, caustic wash from cyclohexane oxidation processes, or wastestream from a chemical or petrochemical industry.

Methods described herein can be performed using isolated enzymes.

Methods described herein can be performed using cell lysates comprisingthe enzymes.

Methods described herein can be performed in a non-naturally occurringhost, such as a recombinant host. For example, the host can be aprokaryote selected from the group consisting of the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as ClostridiumIjungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens or Pseudomonas putida; from the genus Bacillus such asBacillus subtillis; or from the genus Rhodococcus such as Rhodococcusequi. The host can be a eukaryote, for example a eukaryote selected fromthe group consisting of the genus Aspergillus such as Aspergillus niger,from the genus Saccharomyces such as Saccharomyces cerevisiae; from thegenus Pichia such as Pichia pastoris; from the genus Yarrowia such asYarrowia lipolytica; from the genus Issatchenkia such as Issatchenkiaorientalis; from the genus Debaryomyces such as Debaryomyces hansenii;from the genus Arxula such as Arxula adeninivorans; or from the genusKluyveromyces such as Kluyveromyces lactis. The host can be aprokaryotic or eukaryotic chemolithotroph.

The host can be subjected to a fermentation strategy entailinganaerobic, micro-aerobic or aerobic cultivation. A cell retentionstrategy using a ceramic hollow fiber membrane can be employed toachieve and maintain a high cell density during fermentation.

The principal carbon source fed to the fermentation can derive from abiological or a non-biological feedstock. The biological feedstock canbe, or can derive from, monosaccharides, disaccharides, hemicellulosesuch as levulinic acid and furfural, cellulose, lignocellulose, lignin,triglycerides such as glycerol and fatty acids, agricultural waste ormunicipal waste. The non-biological feedstock can be, or can derivefrom, either natural gas, syngas, CO₂/H₂, methanol, ethanol,non-volatile residue (NVR), caustic wash from cyclohexane oxidationprocesses or other waste stream from either the chemical orpetrochemical industries.

The reactions of the pathways described herein can be performed in oneor more cell (e.g., host cell) strains (a) naturally expressing one ormore relevant enzymes, (b) genetically engineered to express one or morerelevant enzymes, or (c) naturally expressing one or more relevantenzymes and genetically engineered to express one or more relevantenzymes. Alternatively, relevant enzymes can be extracted from any ofthe above types of host cells and used in a purified or semi-purifiedform. Extracted enzymes can optionally be immobilized to a solidsubstrate such as the floors and/or walls of appropriate reactionvessels. Moreover, such extracts include lysates (e.g., cell lysates)that can be used as sources of relevant enzymes. In the methods providedby this application, all the steps can be performed in cells (e.g., hostcells), all the steps can be performed using extracted enzymes, or someof the steps can be performed in cells and others can be performed usingextracted enzymes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application pertains. Although methods andmaterials similar or equivalent to those described herein can be used topractice the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdcfinitions, will control. Iii addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and the drawings, andfrom the claims. The word “comprising” in the claims may be replaced by“consisting essentially of” or with “consisting of,” according tostandard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of an exemplary biochemical pathway leading to3-HMG from 4-methyl-2-oxopentanoate.

FIG. 2 is a schematic of an exemplary biochemical pathway leading toisoprene from 3-HMG via the mevalonate pathway.

FIG. 3 contains the amino acid sequences of enzymes which may be usedfor biosynthesizing isoprene from 3-HMG via the mevalonate pathway.

FIG. 4 contains nucleic acid sequences encoding enzymes which may beused for biosynthesizing isoprene from 3-HMG via the mevalonate pathway.

DETAILED DESCRIPTION

In one aspect are provided enzymes and non-naturally occurring, forexample recombinant, host microorganisms for synthesis of 3-HMG from4-methyl-2-oxopentanoate, and/or intermediates thereof, in one or moreenzymatic steps.

In one aspect are provided enzymes and non-naturally occurring, forexample recombinant, host microorganisms for synthesis of isoprene from4-methyl-2-oxopentanoate, and/or intermediates thereof, via a 3-HMGintermediate in one or more enzymatic steps.

In one aspect are provided enzymes and non-naturally occurringrecombinant host microorganisms for synthesis of 3-HMG from4-methyl-2-oxopentanoate, and/or intermediates, in one or more enzymaticsteps comprising use of one or more of a 4-methyl-2-oxopentanoatedehydrogenase, a 3-methylbutanoyl-CoA oxidoreductase, a3-methylbut-2-enoyl-CoA carboxylase, and a 3-methylglutaconyl-CoAhydratase; or using non-naturally occurring host cells expressing one ormore such enzymes. In a further aspect are provided enzymes andnon-naturally occurring recombinant host microorganisms for synthesis of3-HMG from 4-methyl-2-oxopentanoate, and/or intermediates, in one ormore enzymatic steps comprising use of one or more of a4-methyl-2-oxopentanoate decarboxylase, a 3-methylbutanal dehydrogenase,a 3-methylbutanoate-CoA ligase; a 3-methylbutanoyl-CoA oxidoreductase, a3-methylbut-2-enoyl-CoA carboxylase, and a 3-methylglutaconyl-CoAhydratase; or using non-naturally occurring host cells expressing one ormore such enzymes.

In one aspect are provided enzymes and non-naturally occurringrecombinant host microorganisms for synthesis of isoprene and/orintermediates thereof via a 3-HMG intermediate in one or more enzymaticsteps comprising use of one or more of a 4-methyl-2-oxopentanoatedehydrogenase, a 3-methylbutanoyl-CoA oxidoreductase, a3-methylbut-2-enoyl-CoA carboxylase, a 3-methylglutaconyl-CoA hydratase,a hydroxymethylglutaryl Co-A reductase, a mevalonate-kinase, aphosphomevalonate kinase, a diphosphomevalonate decarboxylase, anisopentenyl diphosphate isomerase, and an isoprene synthase; or usingnon-naturally occurring host cells expressing one or more such enzymes.In a further aspect are provided enzymes and non. naturally occurringrecombinant host microorganisms tor synthesis of isoprene and/orintermediates thereof via a 3-HMG intermediate in one or more enzymaticsteps comprising use of one or more of a 4-methyl-2-oxopentanoatedecarboxylase, a 3-methylbutanal dehydrogenase, a 3-methylbutanoate-CoAligase, a 3-methylbutanoyl-CoA oxidoreductase, a 3-methylbut-2-enoyl-CoAcarboxylase, a 3-methylglutaconyl-CoA hydratase, a hydroxymethylglutarylCo-A reductase, a mevalonate-kinase, a phosphomevalonate kinase, adiphosphomevalonate decarboxylase, an isopentenyl diphosphate isomerase,and an isoprene synthase; or using non-naturally occurring host cellsexpressing one or more such enzymes.

Host microorganisms described herein can include pathways that can bemanipulated such that isoprene or its intermediates can be produced. Inan endogenous pathway, the host microorganism naturally expresses all ofthe enzymes catalyzing the reactions within the pathway. A hostmicroorganism containing an engineered pathway does not naturallyexpress all of the enzymes catalyzing the reactions within the pathwaybut has been engineered such that all of the enzymes within the pathwayare expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,gDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once thatchromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or othercompoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

For example, depending on the host and the compounds produced by thehost, one or more of the following enzymes may be expressed in the host:a 4-methyl-2-oxopentanoate dehydrogenase, a 4-methyl-2-oxopentanoatedecarboxylase, a 3-methylbutanal dehydrogenase, a 3-methylbutanoate-CoAligase, a 3-methylbutanoyl-CoA oxidoreductase, a 3-methylbut-2-enoyl-CoAcarboxylase, a 3-methylglutaconyl-CoA hydratase, a hydroxymethylglutarylCo-A reductase, a mevalonate-kinase, a phosphomevalonate kinase, adiphosphomevalonate decarboxylase, an isopentenyl diphosphate isomerase,and an isoprene synthase.

As used herein, the term “mevalonate pathway” refers to a pathway forsynthesis of isoprene comprising enzymatically converting3-hydroxy-3-methylglutaryl-CoA to (R)-mevalonate using ahydroxymethylglutaryl Co-A red uctase; enzymatically converting(R)-mevalonate to (R)-5-phosphomevalonate using a mevalonate-kinaseenzyme; enzymatically converting (R)-5-phosphomevalonate to(R)-5-diphosphomevalonate using a phosphomevalonate kinase enzyme;enzymatically converting (R)-5-diphosphomevalonate to isopentenyldiphosphate using a diphosphomevalonate decarboxylase enzyme;enzymatically converting isopentenyl diphosphate to dimethylallyldiphosphate using an isopentenyl diphosphate isomerase; andenzymatically converting dimethylallyl diphosphate to isoprene using anisoprene synthase enzyme.

In one embodiment the 4-methyl-2-oxopentanoate dehydrogenase is the geneproduct of aceD. In one embodiment the 4-methyl-2-oxopentanoatedehydrogenase is the gene product of citL. In one embodiment the4-methyl-2-oxopentanoate dehydrogenase is classified under EC 1.2.1.-.In one embodiment the 4-methyl-2-oxopentanoatc dehydrogenase has theactivity of an enzyme classified under EC 1.2.1.-. In one embodiment the4-methyl-2-oxopentanoate dehydrogenase is classified under EC 1.2.7.7.In one embodiment the 4-methyl-2-oxopentanoate dehydrogenase has theactivity of an enzyme classified under EC 1.2.7.7.

In one embodiment the 4-methyl-2-oxopentanoate decarboxylase is the geneproduct of ipdC. In one embodiment the 4-methyl-2-oxopentanoatedecarboxylase is classified under EC 4.1.1.74. In one embodiment the4-methyl-2-oxopentanoate decarboxylase has the activity of an enzymeclassified under EC 4.1.1.74. In one embodiment the4-methyl-2-oxopentanoate decarboxylase is classified under EC 4.1.1.43.In one embodiment the 4-methyl-2-oxopentanoate decarboxylase has theactivity of an enzyme classified under EC 4.1.1.43.

In one embodiment the 3-methylbutanal dehydrogenase is the gene productof padA. In one embodiment the 3-methylbutanal dehydrogenase isclassified under EC 1.2.1.39. In one embodiment the 3-methylbutanaldehydrogenase has the activity of an enzyme classified under EC1.2.1.39. In one embodiment the 3-methylbutanal dehydrogenase isclassified under EC 1.2.1.5. In one embodiment the 3-methylbutanaldehydrogenase has the activity of an enzyme classified under EC 1.2.1.5.

In one embodiment the 3-methylbutanoate-CoA ligase is classified underEC 6.2.1.-. In one embodiment the 3-methylbutanoate-CoA ligase isclassified under EC 6.2.1.2.

In one embodiment the 3-methylbutanoyl-CoA oxidoreductase is the geneproduct of IiuA. In one embodiment 3-methylbutanoyl-CoA oxidoreductaseis classified under EC 1.3.8.4.

In one embodiment the 3-methylbut-2-enoyl-CoA carboxylase is classifiedunder EC 6.4.1.4.

In one embodiment the 3-methylglutaconyl-CoA hydratase is classifiedunder EC 4.2.1.18.

In one embodiment the hydroxymethylglutaryl Co-A reductase is the geneproduct of mvaA. In one embodiment the hydroxymethylglutaryl Co-Areductase is classified under EC 1.1.1.34. In one embodiment thehydroxymethylglutaryl Co-A reductase is a Staphylococcus aureushydroxymethylglutaryl Co-A reductase (Genbank Accession No. BAB58707.1,SEQ ID No: 1). See FIG. 3. In one embodiment the hydroxymethylglutarylCo-A reductase is a Staphylococcus aureus hydroxymethylglutaryl Co-Areductase encoded by a nucleic acid having the sequence set forth in SEQID No: 8. See FIG. 4.

In one embodiment the mevalonate-kinase is the gene product of mvak1. Inone embodiment the mevalonate-kinase is classified under EC 2.7.1.36. Inone embodiment the mevalonate-kinase is a Staphylococcus aureusmevalonate-kinase (Genbank Accession No. BAB56752.1, SEQ ID No: 2). SeeFIG. 3. In one embodiment the mevalonate-kinase is a Staphylococcusaureus mevalonate-kinase encoded by a nucleic acid having the sequenceset forth in SEQ ID No: 9. See FIG. 4.

In one embodiment the phosphomevalonate kinase is the gene product ofmvak2. In one embodiment the phosphomevalonate kinase is classifiedunder EC 2.7.4.2. In one embodiment the phosphomevalonate kinase is aStaphylococcus aureus phosphomevalonate kinase (Genbank Accession No.BAB50754.1, SEQ ID No: 3). See FIG. 3. In one embodiment thephosphomevalonate kinase is a Staphylococcus aureus phosphomevalonatekinase encoded by a nucleic acid having the sequence set forth in SEQ IDNo: 10. See FIG. 4.

In one embodiment the diphosphomevalonate decarboxylase is the geneproduct of Mdd. In one embodiment the diphosphomevalonate decarboxylaseis classified under EC 4.1.1.33. In one embodiment thediphosphomevalonate decarboxylase is a Streptococcus pneumoniaediphosphomevalonate decarboxylase (Genbank Accession No. AAK99143.1, SEQID No: 4). See FIG. 3. In one embodiment the diphosphomevalonatedecarboxylase is a Staphylococcus epidermidis mevalonate diphosphatedecarboxylase (Genbank Accession No. AAG02436.1, SEQ ID No: 5). See FIG.3. In one embodiment the diphosphomevalonate decarboxylase is aStreptococcus pneumoniae diphosphomevalonate decarboxylase encoded by anucleic acid having the sequence set forth in SEQ ID No: 11. See FIG. 4.

In one embodiment the isopentenyl diphosphate isomerase is the geneproduct of idi. In one embodiment the isopentenyl diphosphate isomeraseis classified under EC 5.3.3.2. In one embodiment the isopentenyldiphosphate isomerase is a Burkholderia multivorans isopentenyldiphosphate isomerase (Genbank Accession No. ABX19602.1, SEQ ID No: 6).See FIG. 3. In one embodiment the isopentenyl diphosphate isomerase is aBurkholderia multivorans isopentenyl diphosphate isomerase encoded by anucleic acid having the sequence set forth in SEQ ID No: 12. See FIG. 4.

In one embodiment the isoprene synthase is the gene product of ispS. Inone emhndiment the isoprene synthase is classified under EC 4.2.3.27. Inone embodiment the isoprene synthase is a Mucuna pruriens isoprenesynthase (SEQ ID No: 7). See FIG. 3. In one embodiment the isoprenesynthase is classified under EC 4.2.3.27. In one embodiment the isoprenesynthase is a Mucuna pruriens isoprene synthase encoded by a nucleicacid having the sequence set forth in SEQ ID No: 13. See FIG. 4.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species, or can be from multiple sources, i.e., differentspecies. Nucleic acids encoding the enzymes described herein have beenidentified from various organisms and are readily available in publiclyavailable databases such as GenBank or EMBL.

Any of the enzymes described herein that can be used for 3-HMGproduction and/or isoprene production can have at least 70% sequenceidentity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, or 100%) to the amino acid sequence of the corresponding wild-typeenzyme.

For example, a hydroxymethylglutaryl Co-A reductase described herein canhave at least 70% sequence identity (homology) (e.g., at least 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aStaphylococcus aureus hydroxymethylglutaryl Co-A reductase (GenbankAccession No. BAB58707.1, SEQ ID No: 1). See FIG. 3.

For example, a mevalonate-kinase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Staphylococcusaureus mevalonate-kinase. (Genbank Accession No. BAB56752.1, SEQ ID No:2). See FIG. 3.

For example, a phosphomevalonate kinase described herein can have atleast 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aStaphylococcus aureus phosphomevalonate kinase (Genbank Accession No.BAB56754.1, SEQ ID No: 3). See FIG. 3.

For example, a diphosphomevalonate decarboxylase described herein canhave at least 70% sequence identity (homology) (e.g., at least 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aStreptococcus pneumoniae diphosphomevalonate decarboxylase (GenbankAccession No. AAK99143.1, SEQ ID No: 4), or a Staphylococcus epidermidismevalonate diphosphate decarboxylase (Genbank Accession No. AAG02436.1,SEQ ID No: 5). See FIG. 3.

For example, an isopentenyl diphosphate isomerase described herein canhave at least 70% sequence identity (homology) (e.g., at least 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aBurkholderia multivorans isopentenyl diphosphate isomerase (GenbankAccession No. ABX19602.1, SEQ ID No: 6). See FIG. 3.

For example, an isoprene synthase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Mucuna pruriensisoprene synthase (SEQ ID No: 7). See FIG. 3.

The percent identity (homology) between two amino acid sequences can bedetermined by any method known to thosc skilled in the art. In oneembodiment, the percent identity (homology) can be determined byaligning the amino acid sequences using the BLAST 2 Sequences (B 12seq)program from the stand-alone version of BLASTZ containing BLASTP version2.0.14. This standalone version of BLASTZ can be obtained from the U.S.government's National Center for Biotechnology Information web site(www.ncbi.nlm.nih.gov). Instructions explaining how to use the B12seqprogram can be found in the readme file accompanying BLASTZ. B12seqperforms a comparison between two amino acid sequences using the BLASTPalgorithm. To compare two amino acid sequences, the options of B 12seqare set as follows: -i is set to a file containing the first amino acidsequence to be compared (e.g., C:\seql.txt); -j is set to a filecontaining the second amino acid sequence to be compared (e.g.,C:\seq2.txt); -pis set to blastp; -o is set to any desired file name(e.g., C:\output.txt); and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison between two amino acid sequences:C:\B12seq c:\seql.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If thetwo compared sequences share homology (identity), then the designatedoutput file will present those regions of homology as aligned sequences.If the two compared sequences do not share homology (identity), then thedesignated output file will not present aligned sequences. Similarprocedures can be used for nucleic acid sequences except that blastn isused.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptidc aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

This application also provides (i) functional variants of the enzymesused in the methods of the application and (ii) functional variants ofthe functional fragments described above. Functional variants of theenzymes and functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods described herein. The term “functional fragment” asused herein refers to a peptide fragment of a protein that has at least25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%;98%; 99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG, polyhistidine (e.g., hexahistidine (SEQ ID No:14)), hemagluttanin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Hosts can naturally express none or some (e.g., one or more, two ormore, three or more, four or more, five or more, or six or more) of theenzymes of the pathways described herein. Endogenous genes of therecombinant hosts also can be disrupted to prevent the formation ofundesirable metabolites or prevent the loss of intermediates in thepathway through other enzymes acting on such intermediates. Recombinanthosts can be referred to as recombinant host cells, non-naturallyoccurring host cells, engineered cells, or engineered hosts. Thus, asdescribed herein, recombinant hosts can include nucleic acids encodingone or more of a decarboxylase, a kinase, a dehydrogenase, amonooxygenase, an acyl [acyl carrier protein (acp)] dehydrogenase, adehydratase, a thioesterase, or a decarboxyating thioesterase asdescribed in more detail below.

In addition, the production of 3-HMG and/or isoprene can be performed invitro using the isolated enzymes described herein, using a lysate (e.g.,a cell lysate) from a host microorganism as a source of the enzymes, orusing a plurality of lysates from different host microorganisms as thesource of the enzymes.

In some embodiments, the enzymes of the pathways described in FIG. 1 andFIG. 2 are the result of enzyme engineering to improve activity orspecificity using the enzyme structure and wild-type residue diversityto inform the rational enzyme design.

In some embodiments, the nucleic acids encoding the enzymes of thepathways described in FIG. 1 and FIG. 2 are introduced into a hostmicroorganism that is either a prokaryote or eukaryote.

Cultivation Strategies

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be a bacterium from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as ClostridiumIjungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetaffidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing isoprene or precursors thereof.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issatchenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadeninivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingisoprene or precursors thereof.

In some embodiments, 3-HMG is biosynthesized in a recombinant host usinga fermentation strategy that can include anaerobic, micro-aerobic oraerobic cultivation of the recombinant host.

In some embodiments, 3-HMG is biosynthesized in a recombinant host usinga fermentation strategy that uses an alternate final electron acceptorto oxygen such as nitrate.

In some embodiments, isoprene is biosynthesized in a recombinant hostusing a fermentation strategy that can include anaerobic, micro-aerobicor aerobic cultivation of the recombinant host.

In some embodiments, isoprene is biosynthesized in a recombinant hostusing a fermentation strategy that uses an alternate final electronacceptor to oxygen such as nitrate.

In some embodiments, a cell retention strategy using, for example,ceramic hollow fiber membranes can be employed to achieve and maintain ahigh cell density during either fed batch or continuous fermentation inthe synthesis of 3-HMG and/or isoprene.

In some embodiments, the biological feedstock can be, can include, orcan derive from, monosaccharides, disaccharides, lignocellulose,hemicellulose, cellulose, lignin, levulinic acid & formic acid,triglycerides, glycerol, fatty acids, agricultural waste, condenseddistillers' solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166, 1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbial. Biotechnol.,2011, 90, 885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 2011,155, 2011, 293-298; Martin and Prather, Journal of Biotechnology, 2009,139, 61 67).

The efficient catabolism of lignin-derived aromatic compounds suchbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Perez-Pantoja et al, FEMSMicrobial. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7), 2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104,155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2), 163-172;Ohashi et al., Journal of Bioscience and Bioengineering, 1999, 87(5),647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22, 1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoic acid,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

The efficient catabolism of methanol has been demonstrated for themethylotropic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133). The efficient catabolism of CO₂ and H₂, which may bederived from natural gas and other chemical and petrochemical sources,has been demonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium Ijungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15), 5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1), 152-156).

In some embodiments, substntially pure cultures of recombinant hostmicroorganisms are provided. As used herein, a “substantially pureculture” of a recombinant host microorganism is a culture of thatmicroorganism in which less than about 40% (i.e., less than about 35%;30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%; 0.01%; 0.001%;0.0001%; or even less) of the total number of viable cells in theculture are viable cells other than the recombinant microorganism, e.g.,bacterial, fungal (including yeast), mycoplasmal, or protozoan cells.The term “about” in this context means that the relevant percentage canbe 15% of the specified percentage above or below the specifiedpercentage. Thus, for example, about 20% can be 17% to 23%. Such aculture of recombinant microorganisms includes the cells and a growth,storage, or transport medium. Media can be liquid, semi-solid (e.g.,gelatinous media), or frozen. The culture includes the cells growing inthe liquid or inion the semi-solid medium or being stored or transportedin a storage or transport medium, including a frozen storage ortransport medium. The cultures are in a culture vessel or storage vesselor substrate (e.g., a culture dish, flask, or tube or a storage vial ortube).

Metabolic Engineering

The present application provides methods involving less than or morethan all the steps described for all the above pathways. Such methodscan involve, for example, one, two, three, four, five, six, seven,eight, nine, ten, or more of such steps. Where less than all the stepsare included in such a method, the first step can be any one of thesteps listed. Furthermore, recombinant hosts described herein caninclude any combination of the above enzymes such that one or more ofthe steps, e.g., one, two, three, four, five, six, seven, eight, nine,ten, or more of such steps, can be performed within a recombinant host.

In addition, this application recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this application recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This application also recognizes that where an enzyme is shown to accepta particular co-factor, such as NADPH, or co-substrate, such as but notlimited to 3-methylglutaconyl-coA, many enzymes are promiscuous in termsof accepting a number of different co-factors or co-substrates incatalyzing a particular enzyme activity. Also, this applicationrecognizes that where enzymes have high specificity for e.g., aparticular co-factor such as NADH, an enzyme with similar or identicalactivity that has high specificity for the co-factor NADPH may be in adifferent enzyme class.

In some embodiments, the enzymes in the pathways outlined herein can bethe result of enzyme engineering via non-direct or rational enzymedesign approaches with aims of improving activity, improvingspecificity, reducing feedback inhibition, reducing repression,improving enzyme solubility, changing stereo-specificity, or changingco-factor specificity.

In some embodiments, the enzymes in the pathways outlined herein can begene dosed, i.e., overexpressed, into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to isoprene.

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to isoprene.

In some embodiments, one or more enzymes from the pathways describedherein, for example, at least one enzyme classified under EC 1.2.1.-, EC1.2.7.7, EC 4.1.1.74, EC 4.1.1.43, EC 1.2.1.39, EC 1.2.15, EC 6.2.1.-,EC 1.3.8.4, EC 6.4.1.4, EC 4.2.1.18, EC 1.1.1.34, EC 2.7.1.36, EC2.7.4.2, EC 4.1.1.33, EC 5.3.3.2, or EC 4.2.3.27, are introduced or genedosed into a host microorganism that utilizes the non-mevalonate or2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid synthesis. Insome embodiments, at least one enzyme having the amino acid sequencelisted in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ IDNo: 59, SEQ ID No: 6, or SEQ ID No: 7 is introduced or gene dosed into ahost microorganism that utilizes the non-mevalonate or2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid synthesis.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of isoprene, a puridine nucleotide transhydrogenase genesuch as UdhA can be overexpressed in the host organism (Brigham et al.,Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of isoprene, a glyceraldehyde-3P-dehydrogenase gene suchas GapN can be overexpressed in the host organism (Brigham et al., 2012,supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of isoprene, a malic enzyme gene such as macA or maeB canbe overexpressed in the host organism (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of isoprene, a glucose-6-phosphate dehydrogenase gene suchas zwf can be overexpressed in the host organism (Lim et al., Journal ofBioscience and Bioengineering, 2002, 93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of isoprene, a fructose 1,6 diphosphatase gene such as fbpcan be overexpressed in the host (Becker et al., Journal ofBiotechnology, 2007, 132, 99-109).

In some embodiments, the efflux of isoprene across the cell membrane tothe extracellular media can be enhanced or amplified by geneticallyengineering structural modifications to the cell membrane or increasingany associated transporter activity for isoprene.

Producing Isoprene Using a Recombinant Host

3-HMG and/or isoprene can be produced by providing a host microorganismand culturing the provided microorganism with a culture mediumcontaining a suitable carbon source as described above. In general, theculture media and/or culture conditions can be such that themicroorganisms grow to an adequate density and produce isopreneefficiently. For large-scale production processes, any method can beused such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2nd Edition, Editors: A. L. Demain andJ. E. Davies, ASM Press; and Principles of Fermentation Technology, P.F. Stanbury and A. Whitaker, Pergamon). In one example, a large tank(e.g., a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that,additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of 3-HMG and/or isoprene. In one example, a substratecomprising CO is provided to a bioreactor comprising one or moremicroorganisms and anaerobically fermenting the substrate to produceisoprene according to methods described in US 2012/0045807. In oneexample, the microorganisms can be used for the production of isopreneby microbial fermentation of a substrate comprising CO according tomethods described in US 2013/0323820.

Once produced, any method can be used to isolate isoprene. For example,isoprene can be recovered from the fermenter off-gas stream as avolatile product as the boiling point of isoprene is 34.1° C. At atypical fermentation temperature of approximately 30° C., isoprene has ahigh vapor pressure and can be stripped by the gas flow rate through thebroth for recovery from the off-gas. Isoprene can be selectivelyadsorbed onto, for example, an adsorbent and separated from the otheroff-gas components. Membrane separation technology may also be employedto separate isoprene from the other off-gas compounds. Isoprene maydesorbed from the adsorbent using, for example, nitrogen and condensedat low temperature and high pressure:

Additional Exemplary Embodiments

In one embodiment, are methods for synthesizing3-hydroxy-3-methylglutaryl-CoA comprising: enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA, for example by using apolypeptide having the activity of an EC 1.2.7.7 or EC 1.2.1.- enzyme;enzymatically converting 3-methylbutanoyl-CoA to3-methylbut-2-enoyl-CoA, for example by using a polypeptide having theactivity of an EC 1.3.8.4 enzyme; enzymatically converting3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl-CoA, for example by usinga polypeptide having the activity of an EC 6.4.1.4 enzyme; andenzymatically converting 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA, for example by using a polypeptidehaving the activity of an EC 4.2.1.18 enzyme.

In one embodiment are methods for synthesizing3-hydroxy-3-methylglutaryl-CoA comprising: enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanal, for example by using apolypeptide having the activity of an EC 4.1.1.74 or EC 4.1.1.43 enzyme;enzymatically converting 3-methylbutanal to 3-methylbutanoate, forexample by using a polypeptide having the activity of an EC 1.2.1.39 orEC 1.2.1.5 enzyme; enzymatically converting 3-methylbutanoate to3-methylbutanoyl-CoA, for example by using a polypeptide having theactivity of an EC 6.2.1.2 enzyme; enzymatically converting3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA, for example by using apolypeptide having the activity of an EC 1.3.8.4 enzyme; enzymaticallyconverting 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl-CoA, forexample by using a polypeptide having the activity of an EC 6.4.1.4enzyme; and enzymatically converting 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA, for example by using a polypeptidehaving the activity of an EC 4.2.1.18 enzyme.

In one embodiment are provided methods for synthesizing3-hydroxy-3-methylglutaryl-CoA comprising: enzymatically converting4-methyl-2-oxopentanoate to 3-methylbut-2-enoyl-CoA hy: (a)enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanal,enzymatically converting 3-methylbutanal to 3-methylbutanoate, andenzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA; (b)enzymatically converting 4-methyl-2-oxopentanoate to3-methylbutanoyl-CoA; or (c) both (a) and (b); enzymatically converting3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA; enzymaticallyconverting 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl-CoA; andenzymatically converting 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA.

In one embodiment are provided methods for synthesizing3-hydroxy-3-methylglutaryl-CoA comprising: enzymatically converting4-methyl-2-oxopentanoate to 3-methylbut-2-enoyl-CoA by: both (a)enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanal,enzymatically converting 3-methylbutanal to 3-methylbutanoate, andenzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA; and(b) enzymatically converting 4-methyl-2-oxopentanoate to3-methylbutanoyl-CoA; enzymatically converting 3-methylbutanoyl-CoA to3-methylbut-2-enoyl-CoA; enzymatically converting3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl-CoA; and enzymaticallyconverting 3-methyl-glutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA.

In one embodiment are provided methods for synthesizing isoprene via amevalonate pathway comprising: synthesizing3-hydroxy-3-methylglutaryl-CoA according to a method described herein;enzymatically converting 3-hydroxy-3-methylglutaryl-CoA to(R)-mevalonate; enzymatically converting (R)-mevalonate to(R)-5-phosphomevalonate; enzymatically converting(R)-5-phosphomevalonate to (R)-5-diphosphomevalonate; enzymaticallyconverting (R)-5-diphosphomevalonate to isopentenyl diphosphate;enzymatically converting isopentenyl diphosphate to dimethylallyldiphnsphate; and enzymatically converting dimethylallyl diphosphate toisoprene.

In one embodiment are provided methods for synthesizing isoprene via amevalonate pathway comprising: synthesizing3-hydroxy-3-methylglutaryl-CoA according to a method described herein;and one or more steps selected from the group consisting of:enzymatically converting 3-hydroxy-3-methylglutaryl-CoA to(R)-mevalonate using a polypeptide having the activity of an EC 1.1.1.34enzyme; enzymatically converting (R)-mevalonate to(R)-5-phosphomevalonate using a polypeptide having the activity of an EC2.7.1.36 enzyme; enzymatically converting (R)-5-phosphomevalonate to(R)-5-diphosphomevalonate using a polypeptide having the activity of anEC 2.7.4.2 enzyme; enzymatically converting (R)-5-diphosphomevalonate toisopentenyl diphosphate using a polypeptide having the activity of an EC4.1.1.33 enzyme; enzymatically converting isopentenyl diphosphate todimethylallyl diphosphate using a polypeptide having the activity of anEC 5.3.3.2 enzyme; and enzymatically converting dimethylallyldiphosphate to isoprene using a polypeptide having the activity of an EC4.2.3.27 enzyme.

In one embodiment is provided a non-naturally occurring host capable ofproducing 3-hydroxy-3-methylglutaryl-CoA, said host comprising: at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 1.2.7.7 or EC 1.2.1.- enzyme; at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 1.3.8.4 enzyme; atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 6.4.1.4 enzyme; and at least one exogenous nucleicacid encoding a polypeptide having the activity of an EC 4.2.1.18enzyme.

In one embodiment is provided a non-naturally occurring host capable ofproducing 3-hydroxy-3-methylglutaryl-CoA, said host comprising: at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 4.1.1.74 or EC 4.1.1.43 enzyme; at least one exogenous nucleicacid encoding a polypeptide having the activity of an EC 1.2.1.39 or EC1.2.1.5 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 6.2.1.2. enzyme; at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 1.3.8.4 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 6.4.1.4 enzyme; and at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 4.2.1.18 enzyme.

In one embodiment is provided a non-naturally occurring host capable ofproducing 3-hydroxy-3-methylglutaryl-CoA, said host comprising: at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 1.2.7.7 or EC 1.2.1.- enzyme; at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 4.1.1.74 or EC4.1.1.43 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme;at least one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 6.2.1.2. enzyme; at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 1.3.8.4 enzyme; atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 6.4.1.4 enzyme; and at least one exogenous nucleicacid encoding a polypeptide having the activity of an EC 4.2.1.18enzyme.

In one embodiment is provided a non-naturally occurring host asdescribed above wherein said host is capable of producing isoprene andcomprises: at least one exogenous nucleic acid encoding a pulypeptldehaving the activity of an EC 1.1.1.34 enzyme; at least one exogenousnucleic acid encoding a polypeptide having the activity of an EC2.7.1.36 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 2.7.4.2 enzyme; at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 4.1.1.33 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 5.3.3.2 enzyme; and at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 4.2.3.27 enzyme.

In one embodiment is provided a non-naturally occurring host capable ofproducing 3-hydroxy-3-methylglutaryl-CoA, said host comprising at leastone of: at least one exogenous nucleic acid encoding a polypeptidehaving the activity of an EC 1.2.7.7 or EC 1.2.1.- enzyme; at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 4.1.1.74 or EC 4.1.1.43 enzyme; at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 1.2.1.39 or EC1.2.1.5 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 6.2.1.2. enzyme; at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 1.3.8.4 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 6.4.1.4 enzyme; and at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 4.2.1.18 enzyme; and said host further comprising at least one of:at least one endogenous enzyme capable of enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA; at least oneendogenous enzyme capable of enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanal; at least one endogenousenzyme capable of enzymatically converting 3 mcthylbutanal to3-methylbutanoate; at least one endogenous enzyme capable ofenzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA; atleast one endogenous enzyme capable of enzymatically converting3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA; at least one endogenousenzyme capable of 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl-CoA;and at least one endogenous enzyme capable of 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA.

In one embodiment is provided a non-naturally occurring host asdescribed above wherein at least one of the exogenous nucleic acids iscontained within a plasmid.

In one embodiment is provided a non-naturally occurring host asdescribed above wherein at least one of the exogenous nucleic acids isintegrated into a chromosome of the host.

In one embodiment is provide a method as described above wherein saidmethod is performed in a recombinant host.

In one embodiment is provide a method as described above wherein atleast one of the enzymatic conversions is performed in a recombinanthost.

In one embodiment the host is a prokaryotic host, for example from thegenus Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas,Bacillus, or Rhodococcus. In one embodiment the host is Cupriavidusnecator.

In one embodiment the host is a eukaryotic host, for example from thegenus Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia,Debaryomyces, Arxula, or Kluyveromyces.

In one embodiment the host is capable of endogenously producing3-hydroxy-3-methylglutaryl-CoA.

In one embodiment the host is capable of endogenously producing isoprenevia a non-mevalonate pathway.

In one embodiment of the methods and hosts described herein, at leastone of the enzymatic conversions comprises gas fermentation within thehost, for example fermentation of gas comprising at least one of naturalgas, syngas, CO₂/H₂, methanol, ethanol, non-volatile residue, causticwash from cyclohexane oxidation processes, or waste stream from achemical or petrochemical industry.

In one embodiment is provided a method for synthesizing3-hydroxy-3-methylglutaryl-CoA comprising culturing a host describedherein in a gas medium.

In one embodiment is provided a method for synthesizing isoprene via themevalonate pathway comprising culturing a host described herein in a gasmedium. In one embodiment the method further comprises recovering theproduced isoprene. In one embodiment, the host performs the enzymaticsynthesis by gas fermentation. In one embodiment, the gas comprises atleast one of natural gas, syngas, CO₂/H₂, methanol, ethanol,non-volatile residue, caustic wash from cyclohexane oxidation processes,or waste stream from a chemical or petrochemical industry.

In one embodiment is provided a composition comprising3-hydroxy-3-methylglutaryl-CoA synthesized by a method described herein.

In one embodiment is provided a composition comprising isoprenesynthesized by a method described herein.

In one embodiment is provided a method for producing bioderived3-hydroxy-3-methylglutaryl-CoA, comprising culturing or growing a hostdescribed herein under conditions and for a sufficient period of time toproduce bioderived 3-hydroxy-3-methylglutaryl-CoA.

In one embodiment is provided a method for producing bioderivedisoprene, comprising culturing or growing a host described herein underconditions and for a sufficient period of time to produce bioderivedisoprene.

In one embodiment is provided bioderived isoprene produced in a hostdescribed herein, wherein said bioderived isoprene has a carbon-12,carbon-13, and carbon-14 isotope ratio that reflects an atmosphericcarbon dioxide uptake source.

In one embodiment is provided a bio-derived, bio-based, orfermentation-derived product comprising: (a) a composition comprising atleast one bio-derived, bio-based, or fermentation-derived compoundprepared (i) using a host described herein, or (ii) according to amethod described herein, or any combination thereof; (b) a bio-derived,bio-based, or fermentation-derived polymer comprising the bio-derived,bio-based, or fermentation-derived composition or compound of (a), orany combination thereof; (c) a bio-derived, bio-based, orfermentation-derived cis-polyisoprene rubber, trans-polyisoprene rubber,or liquid polyisoprene rubber, comprising the bio-derived, bio-based, orfermentation-derived compound or bio-derived, bio-based, orfermentation-derived composition of (a), or any combination thereof orthe bio-derived, bio-based, or fermentation-derived polymer of (b), orany combination thereof; (d) a molded substance obtained by molding thebio-derived, bio-based, or fermentation-derived polymer of (b), or thebio-derived, bio-based, or fermentation-derived resin of (c), or anycombination thereof; (e) a bio derived, bio-based, orfermentation-derived formulation comprising the bio-derived, bio-based,or fermentation-derived composition or compound of (a), bio-derived,bio-based, or fermentation-derived polymer of (b), bio-derived,bio-based, or fermentation-derived resin of (c), or bio-derived,bio-based, or fermentation-derived molded substance of (d), or anycombination thereof; or (f) a bio-derived, bio-based, orfermentation-derived semi-solid or a non-semi-solid stream, comprisingthe bio-derived, bio-based, or fermentation-derived composition orcompound of (a), bio-derived, bio-based, or fermentation-derived polymerof (b); bio-derived, bio-based, or fermentation-derived resin of (c),bio-derived, bio-based, or fermentation-derived formulation of (e), orbio-derived, bio-based, or fermentation-derived molded substance of (d),or any combination thereof.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications are within thescope of the following claims.

1-12. (canceled)
 13. A non-naturally occurring host capable of producing3-hydroxy-3-methylglutaryl-CoA, said host comprising: (a) at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 1.2.7.7 or EC 1.2.1.- enzyme; or (b) at least one exogenous nucleicacid encoding a polypeptide having the activity of an EC 4.1.1.74 or EC4.1.1.43 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme;at least one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 6.2.1.2. enzyme; or (c) both (a) and (b); and at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 1.3.8.4 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 6.4.1.4 enzyme; and at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 4.2.1.18 enzyme.
 14. The host of claim 13, wherein said host iscapable of producing isoprene and comprises: at least one exogenousnucleic acid encoding a polypeptide having the activity of an EC1.1.1.34 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 2.7.1.36 enzyme; at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 2.7.4.2 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 4.1.1.33 enzyme; at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 5.3.3.2 enzyme; and at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 4.2.3.27 enzyme.
 15. Anon-naturally occurring host capable of producing3-hydroxy-3-methylglutaryl-CoA, said host comprising at least one of: atleast one exogenous nucleic acid encoding a polypeptide having theactivity of an EC 1.2.7.7 or EC 1.2.1.- enzyme; at least one exogenousnucleic acid encoding a polypeptide having the activity of an EC4.1.1.74 or EC 4.1.1.43 enzyme; at least one exogenous nucleic acidencoding a polypeptide having the activity of an EC 1.2.1.39 or EC1.2.1.5 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 6.2.1.2. enzyme; at least oneexogenous nucleic acid encoding a polypeptide having the activity of anEC 1.3.8.4 enzyme; at least one exogenous nucleic acid encoding apolypeptide having the activity of an EC 6.4.1.4 enzyme; and at leastone exogenous nucleic acid encoding a polypeptide having the activity ofan EC 4.2.1.18 enzyme; and said host further comprising at least one of:at least one endogenous enzyme capable of enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA; at least oneendogenous enzyme capable of enzymatically converting4-methyl-2-oxopentanoate to 3-methylbutanal; at least one endogenousenzyme capable of enzymatically converting 3-methylbutanal to3-methylbutanoate; at least one endogenous enzyme capable ofenzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA; atleast one endogenous enzyme capable of enzymatically converting3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA; at least one endogenousenzyme capable of 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl-CoA;and at least one endogenous enzyme capable of 3-methyl-glutaconyl-CoA to3-hydroxy-3-methylglutaryl-CoA.
 16. The host of claim 13, wherein atleast one of the exogenous nucleic acids is contained within a plasmid.17. The host of claim 13, wherein at least one of the exogenous nucleicacids is integrated into a chromosome of the host. 18-20. (canceled) 21.The host of claim 13, wherein the host is a prokaryotic host from thegenus Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas,Bacillus, or Rhodococcus.
 22. (canceled)
 23. The host of claim 21,wherein the host is Cupriavidus necator.
 24. (canceled)
 25. The host ofclaim 13, wherein the host is a eukaryotic host from the genusAspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia,Debaryomyces, Arxula, or Kluyveromyces.
 26. The host of claim 13,wherein the host is capable of endogenously producing3-hydroxy-3-methylglutaryl-CoA.
 27. The host of claim 13, wherein thehost is capable of endogenously producing isoprene via a non-mevalonatepathway.
 28. The host of claim 13, wherein at least one of the enzymaticconversions comprises gas fermentation within the host.
 29. The host ofclaim 28, wherein the gas comprises at least one of natural gas, syngas,CO₂/H₂, methanol, ethanol, non-volatile residue, caustic wash fromcyclohexane oxidation processes, or waste stream from a chemical orpetrochemical industry.
 30. The host of claim 29, wherein the gas isCO₂/H₂.
 31. A method for synthesizing 3-hydroxy-3-methylglutaryl-CoAcomprising culturing the host of claim 13 in a gas medium.
 32. A methodfor synthesizing isoprene via the mevalonate pathway comprisingculturing the host of claim 14 in a gas medium.
 33. The method of claim32, further comprising recovering the produced isoprene.
 34. The methodof claim 32, wherein the host performs the enzymatic synthesis by gasfermentation.
 35. The method of claim 34, wherein the gas comprises atleast one of natural gas, syngas, CO₂/H₂, methanol, ethanol,non-volatile residue, caustic wash from cyclohexane oxidation processes,or waste stream from a chemical or petrochemical industry.
 36. Themethod of claim 35, wherein the gas is CO₂/H₂.
 37. A compositioncomprising 3-hydroxy-3-methylglutaryl-CoA synthesized by the method ofclaim
 31. 38. A composition comprising isoprene synthesized by themethod of claim
 32. 39. A method for producing bioderived3-hydroxy-3-methylglutaryl-CoA, comprising culturing or growing a hostaccording to claim 13 under conditions and for a sufficient period oftime to produce bioderived 3-hydroxy-3-methylglutaryl-CoA.
 40. A methodfor producing bioderived isoprene, comprising culturing or growing ahost according to claim 14 under conditions and for a sufficient periodof time to produce bioderived isoprene.
 41. Bioderived isoprene producedin a host according to claim 14, wherein said bioderived isoprene has acarbon-12, carbon-13, and carbon-14 isotope ratio that reflects anatmospheric carbon dioxide uptake source.
 42. A bio-derived, bio-based,or fermentation-derived product comprising: (a) a composition comprisingat least one bio-derived, bio-based, or fermentation-derived compoundprepared (i) using the host of claim 13, (b) a bio-derived, bio-based,or fermentation-derived polymer comprising the bio-derived, bio-based,or fermentation-derived composition or compound of (a), or anycombination thereof, (c) a bio-derived, bio-based, orfermentation-derived cis-polyisoprene rubber, trans-polyisoprene rubber,or liquid polyisoprene rubber, comprising the bio-derived, bio-based, orfermentation-derived compound or bio-derived, bio-based, orfermentation-derived composition of (a), or any combination thereof orthe bio-derived, bio-based, or fermentation-derived polymer of (b), orany combination thereof, (d) a molded substance obtained by molding thebio-derived, bio-based, or fermentation-derived polymer of (b), or thebio-derived, bio-based, or fermentation-derived resin of (c), or anycombination thereof, (e) a bio-derived, bio-based, orfermentation-derived formulation comprising the bio-derived, bio-based,or fermentation-derived composition or compound of (a), bio-derived,bio-based, or fermentation-derived polymer of (b), bio-derived,bio-based, or fermentation-derived resin of (c), or bio-derived,bio-based, or fermentation-derived molded substance of (d), or anycombination thereof, or (f) a bio-derived, bio-based, orfermentation-derived semi-solid or a non-semi-solid stream, comprisingthe bio-derived, bio-based, or fermentation-derived composition orcompound of (a), bio-derived, bio-based, or fermentation-derived polymerof (b), bio-derived, bio-based, or fermentation-derived resin of (c),bio-derived, bio-based, or fermentation-derived formulation of (e), orbio-derived, bio-based, or fermentation-derived molded substance of (d),or any combination thereof.